Methods for testing for and inhibiting the development of Huntington&#39;s disease

ABSTRACT

This invention relates to Huntington&#39;s disease and more specifically to methods for testing and inhibiting the development of Huntington&#39;s disease.

CROSS-REFERENCE

[0001] This is a continuation-in-part of U.S. Provisional ApplicationSerial No. 60/408,184, filed on Sep. 4, 2002, and a continuation-in-partof U.S. Provisional Application Serial No. 60/443,397, filed on Jan. 29,2003.

FIELD OF THE INVENTION

[0002] This invention relates to Huntington's disease and morespecifically to methods for inhibiting the onslaught of the disease inindividuals who test positive for an expanded CAG trinucleotide repeatin the gene that codes for the huntingtin protein.

BACKGROUND OF THE INVENTION

[0003] Huntington's disease is a degenerative, neurological disease thatis almost exclusively inherited from a parent. About 1 in 10,000 people,or total about 30,000 people in the United States currently haveHuntington's disease and about 150,000 are at risk of having inheritedthe disease from a parent. Individuals who are at risk of developingHuntington's disease inherit a gene (HD gene) from a parent that codesfor a protein now known as the huntingtin protein. The HD gene, whosemutation results in Huntington's disease, is located on chromosome 4 andis characterized by an expanded trinucleotide repeat made up ofcytosine, adenine and guanine (CAG). HD is an autosomal dominantdisorder. Specifically the HD gene is located on a non-sex-linkedchromosome, which means that men and women are equally at risk ofinheriting the HD gene and, if the gene is inherited from just one ofeither parent, the inheriting individual will inevitably develop thedisease.

[0004] Whether an individual has inherited the HD gene that leads to thedevelopment of the disease can be determined by a direct gene test usingblood samples. The genetic test counts the number of CAG repeats in theHD gene region. In individuals who do not exhibit the HD gene, thelength of the repeat pattern occurs up to 38 times or less. Inindividuals who inherit the HD gene, the repeat pattern occurs more than38 times. Only about 1-3% of individuals who develop Huntington'sdisease do not inherit the disease from a parent.

[0005] Previously it was inevitable that an individual who carried theHD gene would at some point in their lives develop the disease. HDresults in the genetically programmed degeneration of neurons. Thedisease causes nerve cells in the striatum of the basal ganglia locateddeep within the brain, particularly those in the caudate nuclei and thepallidium, and nerve cells in the cortex of the outer surface of thebrain, to degenerate via programmed cell death (PCD). PCD is a regulatedprocess by which selective gene expression leads to cell death. Thedisease eventually leads to uncontrolled bodily movements, mentaldeterioration and emotional disturbances.

[0006] Previously, there was no known means to treat, prevent orotherwise inhibit the development of the disease in individuals whoeither carry the HD gene or who have already begun developing thedisease. In the latter group, only symptomatic therapies were availableto target the ensuing symptoms resulting from brain degeneration.

SUMMARY OF THE INVENTION

[0007] It is therefore a primary object of this invention to providemethods for inhibiting the development of Huntington's disease.

[0008] It is a further object of this invention to provide methods fordetermining the optimum time in an individual's life time to beginningan appropriate hormone therapy adapted to inhibit the development ofHuntington's disease.

[0009] It is a further object of this invention to provide methods fordetermining the optimum amount of hormone therapy that is adapted toinhibit the development of Huntington's disease.

[0010] A preferred method of the invention for inhibiting thedevelopment of Huntington's disease in an individual who is at risk ofdeveloping the disease, comprises the steps of: determining that theindividual exhibits a trinucleotide repeat pattern, consisting ofcytosine, adenine, and guanine, is of sufficient number to indicate arisk for developing Huntington's disease; establishing that a serumlevel of a preselected hormone in said individual is below normal;administering one or more hormones, selected from a group consisting ofestrogen, testosterone, their respective precursors, and esters ofestrogen, testosterone, and their respective precursors, in amountssufficient to inhibit development of the disease, wherein the individualtypically exhibits an expanded trinucleotide repeat pattern greater than38. The individual may exhibit repeats higher than 38 including expandedtrinucleotide repeat patterns equal to or greater than 43 or even equalto or greater than 63. The individual typically exhibits a huntingtinpolyglutamine protein comprising greater than 38 glutamines. Toestablish that an individual's serum level is below normal, the methodmay include the step of testing the individual's blood sample by asuitable method such as a polymerase chain reaction.

[0011] The method may further comprise the step of predetermining therate at which one or more of said hormones binds to a polyglutaminelocated at an end of said huntingtin polyglutamine protein to determinean optimum time to begin said administering step and said sufficientamount of said one or more hormones, wherein said predetermining stepcomprises the steps of, obtaining one or more samples of a huntingtinpolyglutamine protein with known numbers of glutamines; mixing saidsample with a labeled estradiol source and a buffering solution;measuring the binding affinity of the labeled estradiol source to thehuntingtin polyglutamine protein. The binding affinity is preferablymeasured with a gamma counter and may be equal to or less than about50,000 counts per minute.

[0012] A preferred method for determining the optimum time foradministering a hormone treatment to inhibit the development ofHuntington's disease in an individual who is at risk of developing thedisease, generally comprises the steps of: determining a plurality ofbinding affinities of estradiol to a huntingtin polyglutamine proteinwith known numbers of glutamines; and measuring the serum level ofhormone in said individual to determine if said serum level is belownormal. The affinity may be measured with a gamma counter and typicallyis equal to or less than about 50,000 per minute and may be equal to orless than about 40,000 counts per minute. The mixing step may furthercomprise mixing said labeled hormone source with a buffering solution.

DETAILED DESCRIPTION OF THE PREFERRED METHODS

[0013] The invention features methods for testing for and inhibiting thedevelopment of Huntington's disease in individuals who carry the HDgene. Specifically the methods of the invention utilize balanced hormonetreatment, including estrogen; testosterone; their precursors such asDHEA and progesterone; and their esters to inhibit the onset of HD thatis partially characterized by neural degeneration specific to the spinyneuron in the striatum. The disease, which is characterized by anexpanded CAG-polyglutamine repeat, typically manifests itself withaging. The juvenile form of the disease has extreme polyglutaminetracts, which may, in some cases, exceed the possibility of hormoneprevention. For example, estrogen and testosterone are widely believedto be psychoprotectants and to inhibit other neurodegenerative diseasessuch as Alzheimer's and Parkinson's disease.

[0014] As hormones such as estrogen, testosterone, and their precursorsdecline with age, the protective effect of these hormones is lost andthe HD disease process advances undeterred. Specifically, for example,estrogen declines by 50% in women by approximately 50 years of age and afurther 80% following menopause. Testosterone's and estrogen's, andtheir precursors', effects on brain functions include, but are notnecessarily limited to, stimulation of the growth of the dendriticspines on spiny neurons and regulation of dopomenergic, serotonergic,adrenergic and glutamatergic functions. Testosterone, estrogen, andtheir precursors also increase the synthesis of certain monoamineneurotransmitters, inhibit their degradation, and interact withneurotrophins that stimulate neural growth and survival.

[0015] Animal models of transgenic mice have been developed for themutant HD gene and specifically it is known that these mice that carrythis transgenic gene containing the first exon of the mutant HD gene andhave the identical function as the human mutant HD gene. The brains ofHD patients and animal models of HD have specific abnormalities that arebelieved to be negatively affected by deficiency of testosterone,estrogen, or their precursors. Abnormalities in monoamingericneurotransmission are implicated in the disease process. HD pathogenesissuggests that an early excess of dopaminergic activity causesexcitotoxic cell death that is followed by a dopaminergic deficiency asneuronal death ensues.

[0016] Estrogen's, testosterone's, and their precursors' knownanti-dopaminergic action in the striatum enables them to function earlyin the disease as neuroprotectants. A differential sensitivity toglutamate causes glutamate toxicity that, in turn, affects spiny neuronsin the striatum. Expression of the huntingtin polyglutamine expansion incells exposed to NMDA-type glutamate receptors causes increasedexcitotoxic cell death when compared to control cells. Estrogen,testosterone and/or their precursors act to suppress mRNA levels ofcertain NMDA-glutamate receptors and is thus able to protect neuronsagainst glutamate toxicity.

[0017] Estrogen, testosterone, and/or their precursors mediateneuroprotection in neurons induced to undergo apoptosis. Apoptosis, orprogrammed cell death, is generally used by multicellular organisms toeliminate unnecessary or dangerous cells. Apoptosis is normallyimportant to the development of the brain and nervous system, the immunesystems and various body tissues. However, in the case of HD, apoptosisleads to excessive degeneration of nerve cells. Apoptosis is induced bythe mutant polyglutamine from the huntingtin protein, causing an influxof Ca+ into a cell through the glutamate receptor. Genes are typicallyessential for PCD and, by blocking the expressed gene products, such asthe HD gene product that codes for the huntingtin protein, PCD isinhibited. In this case, estrogen, testosterone and/or their precursorsbind to the polyglutamine located on the end of the huntingtin proteinto prevent the protein from inducing cell death by preventing aggregateaccumulation in the nuclei of these neurons. The ability of estrogen,testosterone, and their precursors to bind to the huntingtin proteindecreases as one ages because the production of estrogen, testosterone,and their precursors decreases with age. Since the length of the CAGpolyglutamate repeat determines the level of hormone needed to renderthis protein ineffective in causing cell death, it is best to begintreatment before ones natural reserves of hormone are depleted with age.The extent of treatment with estrogen, testosterone, and/or theirprecursors will depend on a given individual based on the length of theCAG glutamine repeat in that individual and the stage of the disease.

[0018] Elevated monoamine oxidase (MAO) activity in the brain of HDpatients is believed to be the basis for the depressive symptoms sooften characteristic of HD. Estrogen, testosterone, and their precursorsinhibit MAO activity and have an antidepressant action. Estrogen's,testosterone's, and their precursors' ability to increase cerebral bloodflow is believed to counter the decreased cerebral and caudate bloodflow associated with HD. Estrogen, testosterone, and their precursorsserve as brain protectants in this process.

[0019] Following is a preferred method for determining the rate ofestrogen binding based on the length of the CAG glutamine repeat.Comparable tests can then be used by physicians in a testing regimen inwhich hormone levels, such as estradiol levels, as well as thepoly-glutamine repeat length of the individual are measured to determinethe optimum time to begin therapy.

EXAMPLE

[0020] Glycerol Bacterial stocks of three different CAG repeat lengthscontained within the first exon of Huntingtin and cloned into the PGexvector 2T were used to inoculate overnight cultures of 25 Ml containing50 mg/mL ampicillin. Pgex is a glutathione transferase fusion expressionsystem Amersham Cat#—27-4587-01—. This system allows for the efficientoverproduction of a target protein along with theglutathione-S-transferase as a fusion. These cultures were previouslygrown and DNA preparations of each were made. They were then sequencedusing dye terminator sequencing technique and run on an ABI 377autosequencer. The results show a 23 CAG repeat contained within thefirst exon of Huntingtin labeled Q23. A 47 CAG repeat contained withinthe first exon of Huntingtin labeled Q47 and a 63 CAG repeat containedwithin the first exon of Huntingtin labeled Q63. The bacterial cultureused to make this DNA prep was divided in half. Half of it was used forthe DNA prep and the other half was used to make the glycerol stock usedto inoculate these cultures. After overnight growth at 37 degrees, thecultures were diluted 1:100 (e.g. 1.25 ML into 125 ml of LB containing50 mg/ml of antibiotic ampicillin) and then allowed to continue to growinto middle log phase 1.5 hours.

[0021] At this point, the bacterial cultures were made using 1.0 mMIsopropyl-b-dThiogalactopyranoside (IPTG) by diluting 100 mM IPTG 1:100(1.25 mL of 100 mM IPTG into 125 ml of growth culture representingapproximately 0.6 OD (optical density) at 590 nM using a common lightspectrophotometer). These three cultures were then allowed to growovernight (20 hrs.) at 30 degrees. These cultures were then centrifugedat 600 rpm for 30 minutes to collect the bacteria. The bacterial pelletswere then transferred to a 1.5 mL conical centrifuge tube and 1.0 mL ofice cold PBS (phosphate buffed saline) was added and the pelletsdissolved by vortexing for five minutes. This was then split into two1.5 mL conical tubes containing 700 uL each for all three bacterialpreps and then 350 uL of 3% N-lauyl sacosine was added to each of thesix tubes, mixed, and then each tube was sonicated for a total of 30seconds using a Fisher probe sonicator Model number F-50. Next the sixtubes were centrifuged in a microfuge top speed for 10 minutes.

[0022] The bacterial supernatant of a rich brown color was addeddirectly to the equivalent of 400 uL of Glutathione Sepharose 4B (strictaffinity for the glutathione portion of the fusion—target protein overproduced) beads washed with ten bead volumes of ice cold PBS. Thebacterial supernatant, as a slurry with the Glutathione sepharose, wasallowed to gently rock for 30 minutes. After the binding was complete,the bead slurry was centrifuged at 200 rpm 20 seconds to pellet the beadand then repetitively washed five times with two bead volumes with icecold PBS. After the last supernatant was removed and discarded, thebeads were subjected to elution with 10 mM reduced glutathione in 50 mMtris Ph 8.0 by allowing incubation for 10 minutes under gentle shaking.These were then centrifuged 30 seconds at 200 rpm and the elute removedand saved to a new tube. Next, 20 ul of protease inhibitor was addedPMSF. Then 30 ul of these were then subjected to polyacylamide gelelectrophoresis (PAGE) 12% gels overnight at 90 volts constant voltage.

[0023] The next day the gels were stained with coomassie brilliant bluein 50% methanol and 10% acetic acid, for two hours at 50 degrees, andthen further destained in the same solution composition minus thecoomassie blue for up to four hours. The resultant image showspurification of all three mutant huntingtin protein bands.

[0024] These pure proteins were then used in the following bindingprotocol. Oestradiol-6-(0-carboxymethyl)oximino-(2-[125I] iodohistaminewas used as the estradiol source. Buffer B preferably comprises: 250 mMtris, 150 mM NaCl, and a Dextran Charcoal solution comprising 0.05%Dextran coated charcoal in buffer B (sigma Cat#6197). About 0.5 ml ofbuffer B was preferably used per reaction. A total of 5 ml of Buffer Bwas aliquoted into a glass borosilicate tube (Fisher Cat# 14-961-26) and10 ul of Oestradiol (I 125) was added containing approximately 2,000,000cpm. About 0.5 ml of this solution was then distributed into eightlabled glass tubes (cat # 14-961-260). The entire tube #1's 500 uL wascounted. Tube #2 through #8 were treated in the following manner:

[0025] Tube#1 Total cpm in 0.5 mL of cocktail.

[0026] Tube #2 zero/0.5 mL cocktail

[0027] Tube #3 Q23/0.5 mL cocktail +50 ul Eluate 1

[0028] Tube #4 Q23/0.5 mL cocktail +50 ul Eluate 1

[0029] Tube #5 Q47/0.5 mL cocktail +50 ul Eluate 3

[0030] Tube #6 Q47/0.5 mL cocktail +50 ul Eluate 3

[0031] Tube #7 Q63/0.5 mL cocktail +50 ul Eluate 5

[0032] Tube #8 Q63/0.5 mL cocktail +50 ul Eluate 5

[0033] Tubes #2-#8 were incubated for 1 hour at room temperature, placedon ice for ten minutes, and 200 uL of the Dextran charcoal solution inbuffer B was added. These tubes were then placed on ice ten minutes andthen centrifuged 15 minutes at 2200 rpm in a Sorvall RC-3 refrigeratedcentrifuge with swinging bucket rotor (Sorvall HL-8) at 4 degrees. Theentire 700 uL supernatant was removed and counted using Genesysmulti-well gamma counter (Laboratories Technology, Inc.) The results aresummarized below:

[0034] Counts Per Minute (CPM): Total: 184,972 (I 125) estradiol inbuffer B (count entire tube) Zero: 27,388 Charcoal Filtered, countsupernatant only Sample HD 50,107 +HD Q23, Charcoal Filtered, Count Sup.Q23: Sample HD 53,797 +HD Q23, Charcoal Filtered, Count Sup. Q23: SampleRD 47,454 +HD Q47, Charcoal Filtered, Count Sup. Q47: Sample HD 44,767+HD Q47, Charcoal Filtered, Count Sup Q47: Sample HD 38,221 +HD Q63,Charcoal Filtered, Count Sup. Q63: Sample HD 38,040 +HD Q63, CharcoalFiltered, Count Sup. Q63:

[0035] The significance of these data is that an affinity for estradiolhas been demonstrated using the mutant form of huntingtin, the causativeprotein in Huntington's disease. It is therefore now applicable thatHuntington Disease patients and their at-risk family members should havetheir polyglutamine repeat length tested and if recognized, to start ona regimen of estradiol, or related hormone, replacement therapy as soonas their normal level of these hormones drop below physiologic levels.Physicians should set up a testing regimen that would determine bothestradiol levels as well as the poly-glutamine repeat length todetermine the optimum time to begin therapy. It is thereby shown thatestradiol is the limiting factor in the progression of Huntington'sdisease. The estradiol level, or the serum level of other applicablehormones, in conjunction with the known CAG repeat length of affectedindividuals, is therefore predictive of the symptomatic onset ofHuntington's Disease and thus will establish the optimum time in theindividual's life to begin administering the appropriate hormonetherapy.

[0036] Although specific features of the invention are described inconnection with some of the preferred methods and not others, this isfor convenience only as some feature may be combined with any or all ofthe other features in accordance with the invention.

[0037] Other embodiments will occur to those skilled in the art and arewithin the following claims:

What is claimed is:
 1. A method for inhibiting the development ofHuntington's disease in an individual who is at risk of developing thedisease, comprising the steps of: determining that the individualexhibits a trinucleotide repeat pattern, consisting of cytosine,adenine, and guanine, is of a sufficient number to indicate a risk fordeveloping Huntington's disease; establishing that a serum level of apreselected hormone in said individual is below normal; administeringone or more hormones, selected from a group consisting of estrogen,testosterone, their respective precursors, and esters of estrogen,testosterone, and their respective precursors, in amounts sufficient toinhibit development of the disease.
 2. The method of claim 1, whereinsaid individual exhibits an expanded trinucleotide repeat patterngreater than
 38. 3. The method of claim 1, wherein said individualexhibits an expanded trinucleotide repeat pattern equal to or greaterthan
 43. 4. The method claim 1, wherein said trinucleotide repeatpattern is equal to or greater than
 63. 5. The method of claim 1,wherein said individual exhibits a huntingtin polyglutamine proteincomprising greater than 38 glutamines.
 6. The method of claim 1, furthercomprising the step of predetermining the rate at which one or more ofsaid hormones binds to a polyglutamine located at an end of saidhuntingtin polyglutamine protein to determine an optimum time to beginsaid administering step and said sufficient amount of said one or morehormones.
 7. The method of claim 6, wherein said predetermining stepcomprises the steps of, obtaining one or more samples of a huntingtinpolyglutamine protein with known numbers of glutamines; mixing saidsample with a labeled estradiol source and a buffering solution;measuring the binding affinity of the labeled estradiol source to thehuntingtin polyglutamine protein.
 8. The method of claim 6, wherein saidaffinity is measured with a gamma counter and is equal to or less thanabout 50,000 counts per minute.
 9. A method for determining the optimumtime for administering a hormone treatment to inhibit the development ofHuntington's disease in an individual who is at risk of developing thedisease, comprising the steps of: determining a plurality of bindingaffinities of estradiol to a huntingtin polyglutamine protein with knownnumbers of glutamines; and measuring the serum level of hormone in saidindividual to determine if said serum level is below normal.
 10. Themethod of claim 9, wherein said affinity is equal to or less than about50,000 per minute.
 11. The method of claim 9, wherein said affinity isequal to or less than about 40,000 counts per minute.
 12. The method ofclaim 9, wherein said mixing step further comprises mixing said labeledhormone source with a buffering solution.